DNA Topoisomerase II-mediated Interaction of Doxorubicin and

Daunorubicin Congeners with DNA

Annette Bodley, Leroy F. Liu, Mervyn Israel, et al.

Cancer Res 1989;49:5969-5978.

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(CANCER RESEARCH 49, 5969-5978, November 1, 1989]
DNA Topoisomerase II-mediated Interaction of Doxorubicin and Daunorubicin
Congeners with DNA1
Annette Bodley, Leroy F. Liu, Mervyn Israel, Ramakrishnan Seshadri, Yoshihiro Koseki, Fernando C. Giuliani,
Stanley Kirschenbaum, Robert Silber, and Milan Potmesil2
Department of Biological Chemistry [A. B., L. F. L.J, The Johns Hopkins University School of Medicine, Baltimore, Maiyland 21205; Departments of Pharmacology
and Medicinal Chemistry, and Cancer Center [M. I., R. S., Y. K.j, University of Tennessee-Memphis, Memphis, Tennessee 38163; Farmitalia Carlo Erba, Centro
Ricerche [F. C. G.], Ñerviano, Italy; and Departments of Radiology fS. K., M. P.] and Medicine [R. SJ, New York University School of Medicine,
New York, New York 10016
ABSTRACT
Three groups of doxorubicin and daunorubicin analogues, differing by
their substituents on the chromophore and sugar moieties, were used in
this study. The 3'-A'-unsubstituted (Group I), 3'-/V-acyl (Group 2), and
.V-,V-alkyl (Group 3) analogues were tested for: (a) in vivo antitumor
activity and in vitro cytotoxicity; (b) cellular or tissue uptake and meta
bolic conversion; (c) strength of DNA intercalation; and (</) interaction
with DNA topoisomerase II (topo-II). Compounds of Group 1 were
cytotoxic, were strongly intercalative, and, except for those with C-14
side chain substitution, induced the formation of topo-II-DNA cleavable
complexes. As shown previously, esterolysis of C-14-acyl substituents
was required to yield a metabolite which can interact with topo-II in the
purified system. The C-14-substituted compounds of Group 2 and their
C-14-unsubstituted metabolites were cytotoxic. These drugs were weak
intercalators, and the C-14-unsubstituted congeners induced cleavable
complex formation in the purified system, but with reduced potency
relative to doxorubicin. The type of the 3 '-^Y-position substituent deter
mined whether Group 3 analogues were cytotoxic and strong intercala
tors, or less active and nonintercalating. Although C-14-unsubstituted
intercalators of Group 3 did not form cleavable complexes in the purified
system, they were cytotoxic.
The study shows that DNA intercalation is required but not sufficient
for the activity by topo-II-targeted anthracyclines. In addition to the
planar chromophore which is involved in intercalation, two other domains
of the anthracycline molecule are important for the interaction with topo-
II: (a) substitution of the ( -14 position totally inhibits drug activity in
the purified system, but enhances cytotoxicity by aiding drug uptake and
presumably acting on other cellular targets; and (b) substitutions on the
3'-.-V position of the sugar ring can, depending on the nature of the
substituent, inhibit intercalation and/or topo-II-targeting activity. These
findings may provide guidance for the synthesis and development of new
active analogues.
INTRODUCTION
Mammalian DNA topoisomerase II, a nuclear enzyme that
alters the topologica! state of DNA and is essential for cell
replication and viability (reviewed in Refs. 1-3), appears to be
the principal target for several groups of anticancer agents (3-
6). A covalent complex between topoisomerase II and DNA is
an obligatory intermediate in the catalysis of DNA topoisomer-
ization. Stabilization of this complex by natural products of
microbial or plant origin and their analogues presents an initial
event which leads to cell death. The stabilization of the complex
can interfere with vital functions involving DNA replication.
Received 2/1/89; revised 6/29/89; accepted 8/3/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1Supported in part by USPHS Grants CA-37082, CA-37209, CA-11655, and
CA-39662 from the National Cancer Institute, NIH, Department of Health and
Human Services; by Grant CH-348A from the American Cancer Society; and
grants from Farmitalia Carlo Erba and the Marcia Slater Society for Research in
Leukemia.
2 To whom requests for reprints should be addressed, at Department of
Radiology, Laboratory of Experimental Therapy, New York University School
of Medicine, 550 First Avenue, New York, NY 10016.
5969
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This interaction appears to play a substantial role in the cyto
toxicity exerted by anthracyclines. The precise nature of the
process, however, remains obscure.
It has been proposed that the formation of a ternary complex
drug-topoisomerase II-DNA could be a prerequisite for the
stabilization of DNA cleavable complexes (7). It is unclear
whether the intercalative mode of drug-DNA binding or just an
increased concentration of drug molecules around topoisomer
ase II-DNA adducts is essential for this step. Our previous
studies (7) have shown that DXR3 analogues with undetectable
or low DNA binding stabilize the cleavable complex between
the enzyme and DNA; these results thus favor the former
possibility. Understandably, even weak DNA binding, not de
tectable with the methods applied previously, could be impor
tant for the formation of drug-topoisomerase II-DNA ternary
complexes.
In the present study, we have analyzed three groups of DXR
and daunorubicin analogues, which differ by the substitution
pattern on the chromophore as well as on the sugar moiety.
The drugs show a range of intercalative strengths from strong
to undetectable. The study has identified some of the structural
properties of the analogues which are connected with DNA
intercalation and/or topoisomerase II inhibition. It also shows
that several biologically active intercalating agents apparently
do not interact with this enzyme.
MATERIALS AND METHODS
Drugs and Drug Treatments. Table 1 lists the 24 DXR or daunorub
icin analogues included in this study. Of the .V-A'-unsubstituted anthra
cyclines, all except ADI21 and AD268 were prepared in the laboratories
of Farmitalia Carlo Erba as previously described (8-12). AD268 and
S'-A'-acyl- or 3'-jV-alkylanthracyclines were synthesized as reported
earlier (13-17). The preparation of ADI 20 and ADI 21 will be described
elsewhere. All products were purified to homogeneity, the purity was
tested by thin-layer and high-performance liquid chromatography, and
each compound was fully characterized by microchemical analysis as
well as by the IR, UV, and nuclear magnetic resonance spectral prop
erties.
3 The abbreviations used are: DXR. doxorubicin (Adriamycin); AD32. N-
trifluoroacetyladriamycin-14-valerate; AD38. A'-acetyladriamycin: AD41. A'-tri-
fluoroacetyladriamycin: AD92, A'-trifluoroadriamycinol; ADI 15. A'-pentafluo-
ropropionyladriamycin; AD 120. iV-trifluoroacetyl-14-mcthoxydaunorubicin:
AD121, 14-methoxydaunorubicin; AD133, A'-acetyladriamycin-14-valerate;
AD143, A'-trifluoroacetyladriamycin-l4-0-hemiadipatc; AD194, /V-(n-bu-
tyl)adriamycin-14-\alerate; ADI98. iV-benzyladriamycin-14-valerate; AD199,
/V.jV-dimethyladriamycin-14-valerate: AD202. A',A'-di(n-butyl)adriamycin-14-val-
erate: AD206, A;tiV-dibenzyladriamycin-14-valerate; AD268, adriamycin-14-
thiovalerate: AD280. A'.A'-dimethyladriamycin: AD284. A'-(n-butyl)adriamycin:
AD285. A',A'-di(fl-butyl)adriamycin: AD288. A'-benzyladriamycin; AD289, N,N-
dibenzyladriamycin; CE. cloning efficiency; DMSO, dimethyl sulfoxide:
4'deoDXR. 4'-deoxydoxorubicin: 4'd4'IDXR. 4'-deoxy-4'-iododoxorubicin: 4-
dmxDNR. 4-demethoxydaunorubicin; DTT. dithiothreitol: 4'epiDXR. 4'-epi-
doxorubicin: HPLC, high-performance liquid chromatography; ¡.a.,intraarteri-
ally. ID50. median inhibition dose; ILS, increase in life span; IR, infrared; LDgo.
lethal dose at the 90°V
cell kill level: PAB. protein-associated DNA single-strand
breaks; SDS. sodium dodecyl sulfate: 14-thia DXR, 14-thiadoxorubicin; UV,
ultraviolet.
 ANTHRACYCLINES AND TOPOISOMERASE II-DNA INTERACTION

For in vitro study, the compounds were dissolved in saline (0.16 M
NaCl), DMSO, or Diluent 12 (polyethoxylated castor oil:ethanol; Phar
maceutical Resources Branch, Division of Cancer Treatment, National
Cancer Institute) diluted with saline (1:5). Saline or Diluent 12 was
also used for the formulation of drugs for in vivo application.
Murine Tumors. P388 lymphocytic leukemia cells were maintained
by serial i.p. passage in DBA/2 mice. For experimental purposes, IO6
cells were injected i.p. into each male BALB/c x DBA/2 F! (hereafter
called CD2F,) or C57BL/6 x DBA/2 F, (hereafter called B6D2F,)
mouse. The assay procedures were similar with the standard National
Cancer Institute protocols (18). Drug treatments began on Day 1 after
leukemia inoculation and, in some experiments, continued for 4 con
secutive days. Drugs dissolved in saline were injected i.v., and those
formulated in Diluent 12, i.p. The injected volume was adjusted to 0.1
ml/10 g of body weight. In order to verify that tumor responsiveness
did not change from one series of experiments to another, DXR was
used in each series as a positive control. No differences in responses
were detected between the treatments with DXR formulated in Diluent
12 as compared with saline. Treated mice were followed up to 90 days,
and the treatment was evaluated either as cures or as the increase in
life span |% of ILS = 100 x (T/C - 1), where T is the median survival
time of the drug-treated group in days, and C is median survival time
in days for untreated controls].
DBA3S, a H cell lymphoma maintained by serial s.c. transplants in
isogeneic mice of the DBA/2J inbred strain (19), was used for phar
macological studies on Day 7 after the tumor transfer.
Tissue Culture Lines. P388 cells were obtained from inoculated DBA/
2 mice and maintained for two passages of transfer in 25-cm3 plastic
tissue culture flasks (Corning Glass Works, Corning, NY) in RPMI
1964 medium (Grand Island Biological Co., Grand Island, NY), sup
plemented with 10% heat-inactivated fetal calf serum (Flow Laborato
ries, Inc., Rockville, MD), 20 ¿tM2-mercaptoethanol (Sigma Chemical
Co., St. Louis, MO), and antibiotics (100 Mg/ml of streptomycin and
kanamycin, and 100 lU/ml of penicillin). The cultures were grown at
37°Cin a humidified atmosphere of 95% air:5% CO2 (20-22).
Exponentially growing P388 or CCRF-CEM cells (human lympho-
blastic leukemia line) were grown in multiwell tissue culture plates or
in 25-cm3 tissue culture flasks (Corning Glass Works). P388 cells were
cultured under conditions specified earlier, and 5x10* cells/ml were
exposed for 4 h to drugs dissolved in saline (for details of drug uptake
studies, see Table 3). CCRF-CEM cells were kept in Eagle's minimal
essential medium (Grand Island Biological Co.) supplemented with
10% heat-inactivated fetal calf serum (Microbiological Associates, Be-
thesda, MD), 2 n\i glutamine, and 50 Mg/ml of gentamycin (23). Test
compounds were dissolved in saline or DMSO (1% DMSO final
concentration in the assay) and added to the cultures (5 x IO5cells/ml)
in a final drug concentration ranging from 0.01 to 5.0 ¿tM, with half-
log increments, and incubated for 48 h at 37'C in a humidified atmos
phere of 95% air:5% CO2. Experiments were repeated at least twice, in
each instance with saline or DMSO-exposed cultures serving as growth

Table 1 Doxorubicin and daunorubicin analogue (name, abbreviation/code number, and chemical formula)
D_
0 OH
1

\R2Y_/A 1'R4HHHHHHHHR5OHHOCH,SCO(CH2),CH,HOHOHOHMOOOoooooR7OCH,OCH,OCH,OC

'R3
R4R2OHOHOHOHOHHHIR3HHHHHHHHi

analogue/name3'-/V-unsubstituted
Type of no.DXRDNRAD121AD2684-dmxDNR4'epiDXR4'deoDXR4'd4'IDXR0RIHHHHHOHHHnOHC'4-I/CÑ3
code
anthracyclinesDoxorubicin
(Adriamycin)Daunorubicin
(daunomycin)1
4-Methoxydaunorubicin"Adriamycin-
14-thiovalerate*4-Demethoxydaunorubicin4'-Epidoxorubicin4'-Deoxydoxorubicin4'-Deoxy-4'-iododoxorubicinR?Abbreviationor

3 '-/V-Acylanthracyclines
/V-Trifluoroacetyladriamycin-14-valerate' AD32AD COCF,COCF,COCH,COCF,COCH,COCF,COCF,COCF2CF,OCO(CH2),CH,OCH,OCO(CH2
iV-Trifluoroacetyl-14-methoxydaunorubicin' 120AD
/V-Acetyladriamycin-14-valerate' 133AD143AD38AD41AD92AD115HHHHHHHHOHOHOHOHOHOHOHOHHHHHHHHH
/V-Trifluoroacetyladriamycin-14-O-hemi-
adipate*
/V-Acetyladriamycin
/V-Trifluoroacetyladriamycin
/V-Trifluoroacetyladriamycinol
A'-Pcntafluoropropionyladriamycin

3'-/V-Alkylanthracyclines/V-(n-Butyl)adriamycin-
4-valerate'/V-Benzyladriamycin-
1 194AD198AD199AD202AD206AD280AD284AD285AD288AD289HHHHHHHHHHOHOHOHOHOHOHOHOHOHOHn-C4H,CH2C6H,
4-valerate*/V./V-Dimethyladriamycin-
1
4-valerate"/V./V-Difn-butylladriamycin-
1
4-valerate'/V./V-Dibenzyladriamycin-
1
4-valerate'/Vt/V-Dimethyladriamycin;V-(n-Butyl)adriamycinN,/V-Di(n-butyl)adriamycin,V-Benzyladriamycin/V.A'-DibenzyladriamycinAD
1

•¿C-14
position substituted (R5).
5970

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 ANTHRACYCLINES AND TOPOISOMERASE ll-DNA INTERACTION

controls, and DXR-treated cultures as quality controls. Cell counts
were determined by hemocytometer and/or in a Model F Coulter
Counter. The percentage of growth inhibition was determined using
drug-exposed and control untreated cultures and expressed in /jmol as
the ID50 (23).
Mouse LI210 leukemia cells were grown as a suspension culture in
Fisher's medium supplemented with 10% heat-inactivated horse serum
and 1% Pen-Strep (Grand Island Biological Co.). Suspensions of IO6
cells/ml were exposed to various drug concentrations for l h at 37°C
in humidified 92% air:8% CO2. Cytotoxic effects of drugs were deter
mined by cloning the cells in Fisher's medium with 15% heat-inacti
vated horse serum, 1% Pen-Strep, and 0.3% Noble agar (Difco Labo
ratories, Detroit, MI), as described previously (24). Cell colonies were
scored under low-power magnification following 2 to 3 wk of incubation
at 37°C.Each experimental point was done in quadruplicate and
repeated 2 to 5 times. Cell viability was determined from CE. "Surviving
fraction" of a cell population was defined as the ratio of CE of drug-
treated cells to the CE of untreated (control) cells, with the LD90
expressed in (¡mol.
Pharmacological Studies. For studies in hosts without tumors, groups
of male A/J mice or Sprague-Dawley rats were given injections i.v. or
i.a. with a therapeutic dose equivalent of a test drug. Following drug
administration, blood samples were obtained at intervals ranging from
0 to 24 h. Serum or plasma aliquots were extracted with chloro-
form:propanol (3:1, v/v), evaporated to dryness, and stored at —¿20°CThe reaction mixture was extracted once with phenol and once with
pending analysis.
DBA/2J mice, bearing 1-wk-old DBA3S tumors, were given injec
tions i.p. of a single dose of the test drugs dissolved in saline (DXR) or and 5 mM MgCl2, pH 8.3, at 80 V for 20 h (32). A scale was constructed
Diluent 12 (AD32 or AD 143) and sacrificed l h after the injection.
Upon removal, the tumor tissue was blotted dry and weighed, then
homogenized in 9 volumes of Tris-HCI buffer, pH 8.5, with 3% SDS
(w/v), and extracted 3 times with two volumes of ethyl acetate:propanol
(9:1, v/v) (17).
For cellular pharmacology studies, P388, CEM, or LI210 cells in unwinding is proportional to the intercalative binding constant of the
log-phase growth were incubated with the test drug for various time
intervals. At the end of each incubation, cell pellets and media super-
natants were obtained. The intracellular 3'-A'-unsubstituted drugs were
extracted with ethanol-saline, and their concentration, as well as the
concentration of metabolites, was measured spectrofluorometrically
against known standards at the excitation wavelength of 479 nm and
emission of 593 nm. For the rest of the drugs, pelleted cells were
sonicated and extracted with ethyl acetate:propanol (9:1, v/v), and
samples were processed as described above for in vivo studies. Details
of the procedures have been published elsewhere (7).
HPLC Analyses. Thawed tissue and plasma samples were reconsti
tuted in 1 ml of methanol and subjected to complementary reverse-
phase and normal phase HPLC analysis (25, 26). Reverse-phase sepa
ration was done with a ¿j-Bondapak/phenyl column using a dual-pump
solvent delivery system (Waters Associates, Milford, MA). The solvent
system consisted of ammonium formate buffer, 0.05 M, pH 4.0, and
acetonitrile. Normal phase Chromatographie separation was achieved
with a Partisil-10 PAC column (30 cm x 3.9 mm; Whatman, Inc.,
Clifton, NJ) and an eluting solvent system of chloroform versus chlo-
roform:methanol:acetic acid:water mixture (85:15:5:1.5, v/v). Samples
derived from in vitro studies were analyzed only by reverse-phase
chromatography; a phenyl-Radialpak lO-^m column (10 cm x 8 mm)
mounted on a Z-Module radial compression unit was used in conjunc
tion with the reverse-phase solvent system specified earlier. The flow reaction
rate conditions and the monitoring systems were described in recent
reports (7, 27). Eluting peaks were identified by retention times relative EDTA, pH 8.3. The cleavage pattern was visualized by autoradiogra-
to those of authentic standards. Peaks were quantified by reference to
standard extraction curves for each drug or metabolite and corrected
for recovery of internal standards.
DNA Alkaline Elution. The methodology has been described in detail
in previous publications (28-30). In all experiments, L1210 cells labeled RESULTS
with [3H]thymidine (Amersham/Searle Corp., Arlington Heights, IL)
and irradiated with 300 rads at 0°Cusing a 137Csradiator (Atomic
Energy of Canada, Ltd.) were used as internal standards in the elution
(24). Cells un labeled with the isotope, 106/ml, were treated with the
drugs for l h at 37°Cin 92% air:8% CO2 atmosphere. Aliquots
5971
containing IO6 drug-treated cells were mixed with IO5 cells serving as
internal standards and then applied to 2-^m-pore-sized polyvinyl chlo
ride filters (Millipore, Bedford, MA). Cells were washed and lysed, and
the duplicate lysates were incubated at room temperature with 0.2 mg/
ml of proteinase K. The elution proceeded as specified previously (30).
SDS, 0.1%, was added to the eluting solution for proteinase K-treated
lysates. Fractions were collected at 3-h intervals over 15 h. Eluted DNA
was precipitated, and the precipitates were collected on Durapore filters
(pore size, 0.22 ^m; Millipore), using a sample manifold, and dried.
Each filter was incubated with 2 M 3,5-diaminobenzoic acid hydrochlo-
ride (Sigma Chemical Co.) for 45 min at 60°C.The DNA-adduct
fluorescence was measured with an Aminco Bowman spectrofluo-
rometer (xenon lamp, 415-nm excitation wavelength, 500-nm emission
detection). Radioactivity of the samples was then also determined. The
data were analyzed, and the frequency of PAB per IO6nucleotides was
calculated (31).
DNA Unwinding Assay and Relative Strength of DNA Intercalation.
Reaction mixtures (60 >i\each) containing 50 mM Tris-HCI, pH 7.5,
100 mM KC1, 10 mivi MgCU, 0.5 mM DTT, 0.5 mM EDTA, 30 ¿ig/ml
of bovine serum albumin, 1.5 pg of supercoiled pC15 DNA, 1.5 ^g of
relaxed pC15 DNA, 45 ng of calf thymus topoisomerase I, and 0.1,
0.5, 2.5, or 12.5 ¿¿g/ml
of the test drug were incubated at 37°Cfor 30
min. Each reaction was stopped with the addition of 15 /il of a
prewarmed solution (37°C)containing 2.5% SDS and 100 mM EDTA.
chloroform and then precipitated with ethanol. Samples were electro-
phoresed at 4°Cin a 1% agarose gel in 90 mM Tris, 90 mM boric acid,
for the evaluation of DNA intercalative strength of a drug relative to
DXR intercalation. The strength of DXR was set, in arbitrary units,
equal to 7. The number >7 indicates a greater, and <7 a lower, ability
of a drug to intercalate. It was assumed that the unwinding angle for
each compound is the same as that of DXR and that the extent of
drug.
Spectrophotometric Determination of Drug-DNA Binding. The
method applied is described in detail elsewhere (7). The absorbance of
anthracycline solutions was measured at 480 nm in the presence (.1)
and absence of DNA (/40),using an ultraviolet/visible light spectropho-
tometer (Model Lambda 3B; Perkin-Elmer, Norwalk, CT). The ratio
A/AO was taken as a measure of the extent of DNA-drug binding, with
values of 0.55 to 0.6 representing strong and those of 0.96 to 1.00 no
drug-DNA association.
DNA-Topoisomerase II. The enzyme as purified to homogeneity from
HeLa cells by modifications of the published procedure (33).
Assay for Topoisomerase II-mediated DNA Cleavage. The assay was
performed as described previously (7, 34). DNA plasmid PMC41 was
linearized with EcoRl and end labeled at the 3' termini with the large
fragment of Escherichia coli DNA polymerase I in the presence of [a-
32P]dATP and unlabeled dTTP. The test drugs were dissolved in DMSO
and diluted to desired concentrations with the incubation buffer. Cleav
age reaction mixtures (20 n\ each) containing 50 mM Tris, pH 7.5, 100
mM KC1, 10 mM MgCl2, 1 mM ATP, 0.5 HIMDTT, 0.5 mM EDTA, 30
Mg/ml of bovine serum albumin, 20 ng of 3'-end labeled DNA, 5 ng of
purified human topoisomerase II, and various drug concentrations (5-
fold dilution) were incubated for 20 min at 37°C.The reactions were
stopped with the addition of SDS to a final concentration of 1%. Each
mixture was treated with proteinase K, 0.2 mg/ml, for 1 h at
37°C.Reaction products were analyzed by electrophoresis using a 1%
agarose gel in a buffer containing 0.089 M Tris-borate and 0.002 M
phy.
Drug Pharmacology and Antitumor Activity. Structural for
mulas of the test drugs are presented in Table 1. The compounds
are divided into three groups according to the substitution
pattern at the 3'-nitrogen of the daunosamine moiety (Table 1,

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 ANTHRACYCLINES AND TOPOISOMERASE II-DNA INTERACTION

Table 2 Doxorubicin and daunorubià n analogues (in vivo and in vitro activity) bition of CEM cells by the most active 3'-W-acyl analogues
in vivo in requires a 3.5-fold (AD41) to 7.3-fold (ADI 15) higher concen
(cures or % of vitro
(ID50,MM*)L1210/n
vitro (LO«,, tration than that of DXR. In the L1210 system, the difference
MM")3'-(V-unsubslituted
DrugP388" ILS4)CEM
anthracyclinesDXRDNRAD121'AD268'4-dmxDNR4'epiDXR4'deoDXR4'd4'IDXR3
in cell kill is approximately 13- to 28-fold.
Analogues of the third group, 3'-Ar-alkylanthracyclines, in
ILS100%
ILS81% clude biologically effective as well as ineffective drugs. Among
ILS130% the C-14 position substituents, AD 194, AD 198, and AD202
ILS161%
ILS270% are highly effective in vivo against P388 leukemia and in vitro
ILS300% against CEM and L1210 cell lines. AD 199, despite modest in
ILS300%
ILS100% vivo activity, shows marked activity in the in vitro assays. In the
CEM growth inhibition assay, AD 199 is 3-fold more active
'-A/-Ac)
lanthracyclinesAD32'AD120'AD13.VAD
than DXR. AD280 and AD288, the respective C-14-unsubsti-
cures63%
ILS81% tuted analogues of AD 199 and AD 198, are effective in vitro. In
ILS100%
143'AD38AD41AD92AD1I53'-iV-AlkylanthracvclinesAD
vivo, AD280 shows a modest enhancement in antitumor activity
cures27% compared with AD 199, while AD288 is significantly less effec
ILS481%
ILS100% tive than AD198. AD284, a C-14-unsubstituted analogue of
ILS372% AD 194, is almost as effective against CEM leukemia as are
ILS>475% DXR and AD 199. AD289 and its C-14-substituted congener
AD206 remain without major effectiveness against tumors in
194'AD198'AD199'AD202'AD206'AD280AD284AD285AD288AD289320%
ILS100% vivo as well as in vitro. AD206 is metabolic-ally converted into
cures72%
ILS>770% AD289 and AD288 in vivo, but not in vitro.
ILS145% The compounds of the first group are stable in various
ILS164%
ILS85%
biological systems, both in vivo and in vitro (Table 3). The rate
of conversion into a reduced biologically active form (DXN, C-
13-OH 4-dmxDNR, C-13-OH 4'deoDXR, or C-13-OH
ILS75% 4'd4'IDXR) is relatively small. Of the drugs in the second
ILS0.050.030.320.450.0030.030.030.0090.33.572.50.261.20.211.030.440.110.110.022.1>5.30.30.080.40.5>5.00.310.92.00.230.420.320.188.315.28.87.012.37.21
group, the C-14 position-substituted 3'-yV-acyl analogues AD32
°P388. i.p. mouse leukemia; CEM, tissue culture line of a human leukemia;
LI210. a tissue culture line of a mouse leukemia. Results confirmed in at least and AD143 are largely converted into the C-14-unsubstituted
one independent experiment for each test system. congener AD41. This esterolysis has been documented in pre
* % of ILS. increase in life span, optimal drug doses qd 1 (3'-A/-unsubstituted
vious studies (7, 19, 25, 27). Similarly, the C-14-substituted 3'-
drugs except AD268) or qd 1-4 (the rest of the drugs).
' ID50, median inhibition dose, 48-h drug exposure; mean of 2 to 3 experiments yV-alkylanthracyclines AD 198 and AD 199 are partially trans
with a SD <10% of values shown.
'LO«,, a dose killing 90% of clonogen, 1-h drug exposure; mean of 2 to 5
formed into the C-14-unsubstituted products AD288 or AD280,
experiments with a SD < 20% of values shown. respectively. AD280 or AD288 appears stable, and no further
'tl4 position-substituted analogues. biotransformation has been detected in vitro.
Drug Interaction with DNA. The intercalative binding of each
analogue to DNA was measured using an assay in which both
supercoiled and relaxed DNAs are incubated with the drug in
R3 and R4). Except for ADI21 and AD268, the anthracycline the presence of DNA-topoisomerase I and allowed to come to
molecule of the 3'-./V-unsubstituted analogues is modified at an equilibrium. The inclusion of both topological forms of DNA
either the 4'-position of the daunosamine (Rl and R2) or the ensures that any inhibitory effect which the drugs may exert on
C-4 position of the chromophore (R7). AD268 is the only topoisomerase I will be detectable and could be compensated
analogue of this group with a C-14 position bulky acyl side for in the assay conditions (32). Fig. 1 shows some of the gels
chain (R5), whereas ADI21 contains a nonhydrolyzable 14-0- with assayed drug intercalation. Table 4, first data column,
methyl ether function. A second group, the 3'-/V-acyl analogues, presents the results expressed as the relative strength of drug
is characterized by an acyl function attached to the nitrogen of intercalation. The rating was, on a relative scale, from 0 (no
the daunosamine (R4). Four of the analogues included in this intercalation) to 9 (strong intercalation). It appears that the test
group also have C-14 position side chain substitution (R5). The drugs could be divided into three categories, strong, weak, and
analogues of the third group, 3'-./V-alkylanthracyclines, are nonintercalating compounds. With the exception of AD202
modified by mono- or dialkyl substitution at the sugar nitrogen and its C-14-unsubstituted congener AD285, all biologically
active 3'-/V-unsubstituted analogues (first group) and 3'-N-
(R3, or R3 and R4). In some of the compounds, there is also a
valerate side chain substitution at C-14. alkylanthracyclines (third group) are strong intercalators. The
Drug effectiveness was tested in vivo and in vitro, using several two biologically inactive jV-alkyl derivatives AD206 and AD289
systems (Table 2). A single dose of 3'-./V-unsubstituted ana did not show detectable intercalation. Analogues of the second
group (3'-/V-acylanthracyclines) are generally weak intercala
logues on Day 1 improved, to a varying degree (81 to 320% of
ILS), the survival of mice implanted a day earlier with P388 tors, with the relative strength <5. There was no evidence of
leukemia. The drugs were also effective against CEM in vitro. intercalation with AD32. Within the group, there is no apparent
The in vivo treatment of P388 leukemia with the second group correlation between the strength of intercalation and antitumor
of drugs, 3'-/V-acylanthracyclines, was based on an established activity (compare active compounds AD32, AD 143, AD41, and
optimal treatment schedule (every day, Days 1 to 4) in terms of AD115 with the less active AD133, AD38, or AD92).
cures or ILS. Both C-14 position substituted and unsubstituted Fig. 2 compares the relative strength of DNA intercalation
analogues are represented by highly effective (AD32, AD 143, with the extent of DNA-drug binding, as has been determined
AD41, or AD 115) and less potent compounds (AD 120, AD 133, spectrophotometrically. It is apparent that, in general, both
AD38, and AD92). The differential efficacy of the 3'-JV-acyl methods concur: 3'-yV-unsubstituted as well as 3'-TV-alkylan
analogues is further confirmed by the results of the in vitro thracyclines are strong; and 3'-./V-acyl analogues are weak DNA
CEM and LI210 screens. A comparable in vitro growth inhi intercalators or binders. One exception to this generalization is
5972
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 ANTHRACYCLINES AND TOPOISOMERASE M-DNA INTERACTION

Table 3 Doxorubicin and daunorubkin analogues (metabolism in vivo and in vitro)

Biologically ineffective metabolites are not listed.
vivo% vitroMetabolitesDXN»

Drug.V-V-miMihsi lineP388"L1210'P388"P388'P388"P388'P388"CEM'L1210*L1210*CEM'CEM
Unchanged91.979.099.099.085.016.726.753.96.515.0In Unchanged100.0100.0100.096.0100.099.097.085.594.419.280
strainDBA3S-B6DF/C3H'BALBA/B6D2F/A/J*DBA3S"Sprague-Dawley*DBA3S-Sprague-Dawley7In
anthracvclinesDXR4-dmxDNR4'epiDXR4'deoD\R4'd4'IDXR3'-W-AcylanthracvclinesAD32'AD
¡in
teil
(8.1)'C-13-OH(21.0)C-13-OH(1.0)C-13-OH(1.0)C-13-OH(14.0)AD41

(4.0)C-13-OH(1.0)C-I3-OH(3.0)AD41

(81.1)AD92(I.3)AD41 (11.7)AD92
(0.5)DXR
(63.4)AD920.7)DXR (0.4)AD41
(5.6)AD41
143'AD41AD923'-/V-AlkylanthracyclinesAD (4.5)AD41
(42.3)AD920.9)DXR (80.8)DXR

(1.9)AD41
(87.4)AD92(1.6)DXR

(2.0)AD288
(7.2)AD92
(0.6)DXN
(6.0)AD288(15.1)AD280
198«AD199«AD280AD288Mouse/rat (77.0)C-13-OH(8.0)Cell

(9.7)

" DBA3S lymphoma implanted s.c. into DBA/2J mice, levels of the drug and metabolites in the tumor tissue, 6 h after the i.p. injection of DXR ( 15 mg/kg), and
1 h after the injection of AD32 (80 mg/kg) or AD143 (64 mg/kg), 2 to 4 mice/point.
* DXN, doxorubicinol (adriamycinol).
' Numbers in parentheses, percentage.
* P388, mouse leukemia line, 4-h exposure to 2.5 itg/mi, cell-associated drug uptake.
'L1210. mouse leukemia line. 1-h exposure (MM)to DXR (0.88), AD32 (0.56), AD143 (1.0), AD198 (2.35), AD199 (4.1), and AD280 (2.38); cell-associated drug
and metabolite uptake.
7Mice of various strains, levels of the drug, and metabolite in serum following an i.p. bolus (3 to 10 mg/kg).
' C-14 position-substituted analogues.
* A/J mice and Sprague-Dawley rats, levels of the drug, and metabolites at 3 h in serum following an i.v. bolus (5 mice), or in plasma following an ¡.a.bolus (4
rats); the bolus of AD143 was 50 mg/kg and of AD198, 5.0 mg/kg.
' CEM. human leukemia line. 45-min exposure to 5.2 JIM(AD32) or 3-h to 10 MM(AD41 and AD92), cell-associated drug, and metabolite uptake. All tissue culture
experiments done in duplicates.

ABCDEFGHIJKLMNOPQRABCDEFGHIJKLMNO P Q R

ABC

Fig. 1. Drug intercalation. Form I pC15 DNA was relaxed using purified topoisomerase I in the presence of various drug concentrations. /, A. 1.5 ngof supercoiled
and 1.5 ng of relaxed DNA; B, DNA plus 45 ng of topoisomerase I (controls); C to F, DNA plus topoisomerase I plus DXR (0.18, 0.92, 4.6, or 23.0 MM;3'-N-
unsubstituted drug. Group 1); G to J, 4'deoDXR [0.19, 0.95, 4.75, or 23.75 MM( 1)]; K to N, 4'd4'-IDXR [0.15, 0.76, 3.8, or 19.12 MM( 1)]; O to A, 4'epiDXR [0.18,
0.92,4.6, or 23.0 MM(1)]. 2, A and A, controls; CtoF, DXR(l); G to /, AD92 (0.16, 0.78, 3.9, or 19.5 MM;3'-/V-acylanthracycline, Group 2); K to A/, AD41 (0.16,
0.78, 3.9, or 19.5 MM(2)]; O to R, AD38 [0.17, 0.85, 4.27, or 21.37 MM(2)]. 3, A and B, controls; C to F, DXR (1); G to J, ADI 15 [0.14, 0.72, 3.62, or 18.12 MM
(2)]; ATto A', ADI 33 [0.15. 0.74. 3.72. or 18.62 MM(2)]; O to K, AD206 (0.12, 0.62, 3.1, or 15.5 MM;3'-/V-alkylanthracycline, Group 3). 4. A and B. controls; C to F,
DXR (1); G to J, AD198 [0.14, 0.7, 3.47, or 17.37 MM(3)]; K to N, ADI99 [0.15, 0.76, 3.8, or 19.0 MM(3)]; O to A, 4'dmxDNR [0.2, 1.0, 5.02, or 25.12 MM(1)].

5973
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 ANTHRACYCLINES AND TOPOISOMERASE ll-DNA INTERACTION

Table 4 Doxorubicin and daunorubicin analogues (DNA intercalation, topoisomerase ¡l-mediatedDNA cleavage, and inhibition of background cleavage)
Cell-free system
Topoisomerase II-mediated
cleavage
1210 cells
of background (PAB°induced
effect(flM)0.920.21.010.180.190.7721.4W19.519.518.1
cleavage, maximum by 5 f¿\idrug
Drug.V-.V-misiilisiitiiU'u (relativestrength)77679986031243226663076<170Range(fM)0.04-0.920.04-0.95ND<ND0.04-1.010.04-0.920.04-0.950.03-3.80NDNDNDND0.85-21.40.78-19.5
effect
OIM)>4.620.2aO.725.024.6>4.719.1ND18.2wND16.2
concentration)1.410.760.840.171.151.161.162
anthracyclinesDXRDNRAD121»AD268'4-dmxDNR4'epiDXR4'deoDXR4'd4'IDXR3

'-A'-AcylanthracyclinesAD32'AD

120*ADI33*AD143'AD38AD41AD92AD1153'-A/-AlkylanthracyclmesAD

wX).7>0.720.15>0.7IS.Sw20.04>0.0720.0617.2L

w19.1Inhibition

194'AD198»AD

199»AD202'AD206*AD280AD284AD285AD288AD289DNAintercalation

* PAB, protein-associated DNA cleavage, frequency/10' nucleotides; average of 2 to 3 experiments.

* C-14 position substituted.
' ND, not detectable; w, weak.
' Insignificant frequency of PAB.

10- changes the 3'-Ar-unsubstituted and 3'-7V-acyl-inactive ana

•¿
D
O age. Finally, AD202 and its C-14-unsubstituted
0-Lû_
1 0.8 0.6
Spectrophotometnc
Assay
(A/Ao)
Fig. 2. Comparison of DNA-drug association measured by the unwinding and
spectrophotometric assays. For details, see "Materials and Methods" and Ref. 7.
AD 198, which, in the unwinding assay, shows strong interca
lation, whereas little drug-DNA interaction was seen with the
spectroscopic method (16, 35).
Topoisomerase II-mediated DNA Cleavage. Drug induction
of the topoisomerase II-mediated cleavage of plasmid DNA
was tested in cell-free systems. The results are presented in
Table 4, second and third columns, as the range of drug con
centrations at which DNA cleavage was observed and the opti
mal concentration at which maximal response was induced.
Representative gels are shown in Fig. 3. The most striking
feature was the inability of C-14 position-substituted analogues
to inhibit topoisomerase II and induce DNA cleavage. Ten
analogues with three types of C-14 substituents were tested.
The removal of a thiovalerate, valerate, or O-hemiadipate
5974
logues into active inhibitors of topoisomerase II. Such biotrans
formation converts AD268 (first group) into 14-thiaDXR and
its disulfide. For the compounds of the second group, the
metabolic conversion of AD32 or AD143 results in AD41 and
AD92, and of AD133 in ADI 15. There is a notable exception
to this rule among the C-14-unsubstituted 3'-yV-alkylanthracy-
clines. Neither biologically active AD280 and AD288 nor inac
tive AD289 has shown topoisomerase II-mediated DNA cleav
congener
AD285 are weak biologically active intercalators. AD285 is the
only drug among the tested 3'-./V-alkyl analogues which induces
topoisomerase II-mediated cleavage. In terms of DNA interca
lation, both congeners AD202 and AD285 behave more like
analogues of Group 2.
Incubation of the 3'-end-labeled DNA with purified topoi
somerase II in the absence of a drug results in slight DNA
cleavage (Fig. 3, Lane B). This low level of activity has been
termed "background" cleavage (33). As has been shown in
previous studies (7, 34), DXR stimulates topoisomerase II-
mediated cleavage of plasmid DNA at the concentration range
of 0.04 to 0.92 ßM,and at a concentration of 4.6 Õ¿M
and higher
the cleavage reaction is either diminished or totally abolished
(Fig. 3; Table 4). The rest of 3'-./V-unsubstituted DXR ana
logues, except AD268, stimulated the cleavage with comparable
efficacy. The C-14 position-unsubstituted analogues of the 3'-
yV-acylgroup did not show the background inhibition. For such
effects, substantially higher drug concentrations may be re
quired than those used in the assay. AD268, a C-14-substituted
analogue of the first group, and AD 198, AD 199, AD280, or

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 ANTHRACYCLINES AND TOPOISOMERASE II-DNA INTERACTION

ABCOEFGHIJKLMNOPQ ABCDEFGHIJKLMNOPQ

1 2
Fig. 3. Drug-stimulated topoisomerase Il-me-
diated DNA cleavage. /, A, 20 ng of PMC41 DNA
end labeled with (a-32P]dATP; B, DNA plus 5 ng
ABCDEFGHIJKLMNOPQ ABCDEFGHIJKLMNOPQ
of human DNA-topoisomerase II (controls); C to
G, DNA plus topoisomerase II plus DXR (0.04,
0.18, 0.92, 4.6, or 23.0 ¿¡M; S'-yv-unsubstituted
drug. Group 1); H lo L, 4'epiDXR [0.04, 0.18,
0.92, 4.6, or 23.0 UM (1)]; M to Q, 4-dmxDNR
[0.04, 0.2, 1.0, 5.02, or 25.12 MM(1)]. 2, A and B,
controls; C to G, DXR; H to L. 4'd4'IDXR [0.03,
0.15, 0.76, 3.8, or 19.12 MM (I)]; M to Q,
4'deoDXR [0.04, 0.19, 0.95, 4.75, or 23.75 MM
(1)]. 3. A and B, controls; C to G, DXR (1); H to
III
L, AD32 (0.03, 0.14, 0.69, 3.45, or 17.25 MM;3'-
W-acylanthracycline, Group 2); M to Q. AD 143 *.»
[0.03, 0.13, 0.65, 3.25, or 16.25 MM(2)]. 4, A and
A, controls; C to G, DXR (l); H to L, AD41 [0.03,
0.16, 0.78, 3.9, or 19.5 MM(2)]; M to Q, AD92
[0.03, 0.16, 0.78, 3.9, or 19.5 MM(2)]. 5, A and B.
controls; C to G, DXR (1); H to L, AD 198 (0.03,
0.14, 0.7, 3.47, or 17.37 MM;3'-A'-alkylanthracyc-
lines, Group 3); M to Q, AD 199 [0.03, 0.15, 0.76,
3 4
3.8, or 19.0 MM(3)). 6, A and B, controls; C to G,
DXR (1); H to L, AD280 [0.04, 0.17, 0.87, 3.62,
or 18.12 MM(3)]. ABCDEFGHIJKLMNOPQ ABCDEFGHIJKL

AD288, biologically active drugs of the third group, effectively
abolished the background cleavage reaction in the absence of
topoisomerase H-mediated DNA cleavage.
The drug effects on intact cells were tested using the LI210
system. At a 5 ¿IMconcentration, the test drugs induced a
significant number of PAB (Table 4), except for the two biolog
ically inactive 3'-jV-alkylanthracyclines AD206 and AD289.
Among the 3'-./V-alkyl derivatives, both of these analogues are
least cytotoxic and show the lowest number of PAB. Detection
of PAB in cells treated with 3'-7V-acylanthracyclines provided
further evidence that, at cytotoxic concentrations, the prodrugs
(AD32, AD120, AD133, and AD143) are intracellularly con
verted into topoisomerase H-interacting metabolites.
DISCUSSION
The stabilization of the topoisomerase II-DNA cleavable
complex by anthracyclines appears to be a major cause of drug-
5975
exerted cytotoxicity (4-6). The present study indicates that this
conclusion is valid for most but not all DXR analogues. In
addition to the clinically important chemotherapeutic agent
DXR, the test drugs included several potentially useful com
pounds (for review, see Ref. 36). Some of the analogues, such
as 4'epiDXR, dmxDNR, or AD32, have been investigated in
clinical trials; others are being screened in initial or advanced
preclinical tests. In the present study, several differences in the
structure-function relationship have been detected among
closely related congeners. Summary of experimental findings is
presented in Table 5.
DNA Intercalation of Test Drugs. The extent of DNA inter
calation seems to depend on the substitution of the daunosa-
mine nitrogen (Table 1, R3 and R4). The 3'-yV-unsubstituted
analogues are strong intercalators. Analogues bearing one or
two ./V-alkylsubstituents, such as A'-(n-butyl), TV-benzyl,or N,N-

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 ANTHRACYCLINES AND TOPOISOMERASE II-DNA INTERACTION

Table 5 Summary of experimental findings

The semiquantitative evaluation of various activities was as follows: +++. high; ++, intermediate; +, low; 0, absent.
Activity Topoisomerase
DNA II-mediated
Analogue (abbreviation) In vivo In vitro* intercalation" cleavage"
3'-,V-unsubsli(uted anthracyclines
Doxorubicin (DXR)
Daunorubicin (DNR)
14-Methoxydaunorubicin' (ADI 21)
Adriamycin-14-thiovalerate'(AD268)
4-Demethoxydaunorubicin (4-dmxDNR)
4'-Epidoxorubicin (4'epiDXR)
4'-Deoxydoxorubicin (4'deoDXR)
4'-Deoxy-4'-iododoxorubicin(4'd4'IDXR)

S'-A'-Acylanthracyclines
A-Trifluoroacetyladriamycin-14-valerate' (AD32)
A-Trifluoroacetyladriamycin-14-methoxydaunorubicin' (AD 120)
A-Acetyladriamycin-14-valerate' (ADI 33)
A-Trifluoroacetyladriamycin-14-O-hemiadipate'(AD143)
A-Acelyladriamycin (AD38)
A'-Trifluoroacetyladriamycin (AD41 )
A-Trifluoroacetyladriamycinol(AD92)
A'-Pentafluoropropionyladriamycin (ADI 15)

3'-A'-Alkylanthracyclines
Ar-(n-Butyl)adriamycin-14-valerate'(ADI94)
A'-Benzyladriamycin-14-valerate' (AD 198)
A',A'-Dimethyladriamycin-14-valerate'(AD199)
A',A'-Di(fl-butyl)adriamycin-l 4-valerate'(AD202)
A'.A'-Dibenzyladriamycin-l 4-valerate'(AD206)
A'.A'-Dimethyladriamycin (AD280)
A-(n-Butyl)adriamycin (AD284)
AT,A'-Di(n-butyl)adriamycin(AD285)
A'-Benzyladriamycin (AD288)
A'.A'-Dibenzyladriamycin (AD289)
•¿
+ See Table 4.
•¿See
Table 2.
< 14 position-substituted analogues (Table 1).

dimethyl, also intercalate strongly. Their type of association
with DNA could be at variance with that of DXR-DNA binding
(37). W-Dibenzylation, as in AD206 or AD289, and W-dibu-
tylation, as in AD202 or AD285, appear to significantly inter
fere with or preclude DNA interaction sterically.
The 3'-¿V-acylanthracyclines have either yV-acetyl, TV-trifluo-
roacetyl, or /V-pentafluoropropionyl substitution. All analogues
of this group, except for AD32, have shown a weak DNA
intercalation. In this regard, the present results confirmed the
lack of drug-DNA intercalation for AD32, as evidenced by a
variety of other techniques (38-41). The limited sensitivity of
anthracycline-DNA absorbency assay used in previous work (7,
42) has precluded the detection of low-level DNA intercalation
by AD 143, AD41, and AD92. The data for AD32 suggest that
the bulky C-14 substituent may further affect the weakened
DNA binding caused by the S'-A'-trifluoroacetyl substitution.
Drug Effects on Topoisomerase H-mediated DNA Cleavage.
The side chain C-14 position substitution by a thiovalerate
(AD268), valerate (AD32 and AD133), hemiadipate (AD143),
or O-methyl ether (AD 120 and ADI21) prevents the analogues
from interacting with topoisomerase II (Table 4; Fig. 3). The
metabolic cleavage of a hydrolyzable, C-14 position-acyl sub
stituent by plasma esterases, which has been extensively docu
mented for AD32, AD 143, and related drugs in vivo and in
vitro (7, 43), converts AD32 or AD 143 into AD41 and AD92.
Similarly, ADI33 is converted into AD38, while AD268 is
deesterified, presumably to 14-thiaDXR and its disulfide. All
C-14 position-unsubstituted 3'-./V-acylanthracyclines have been
shown to stimulate the topoisomerase 11-mediated cleavage.
Thus, the study confirms previous observations (7) and lends
further support for the conclusion that metabolic activation of
3'-ALacyl C-14 substituents precedes their interaction with to
5976
poisomerase II. This also becomes applicable to the 3'-7V-alkyl
compound AD202. For all active S'-W-unsubstituted and 3'-N-
acylanthracyclines (Groups 1 and 2), the strength of DNA
cleavage paralleled the strength of drug intercalation. The three
3'-jV-alkyl analogues (Group 3) AD280, AD284, and AD288
are C-14-unsubstituted DNA intercalators which, however, do
not stimulate the topoisomerase II mediated DNA cleavage.
Although cytotoxic in vitro, they exhibit only moderate in vivo
antitumor activity.
Inhibition of "Background" DNA Cleavage. Previous studies
(7, 34) have shown that high concentrations of DXR not only
inhibit the drug-induced DNA cleavage but also the background
cleavage. It appears that, among S'-W-unsubstituted and 3'-N-
alkyl analogues, the inhibition of the background cleavage is a
function of the ability of a drug to intercalate into DNA. This
applies to both C-14-substituted and -unsubstituted analogues.
However, AD280, AD284, and AD288, the C-14-unsubstituted
3'-/V-alkylanthracyclines, do not cleave DNA in the presence
of topoisomerase II, yet the drugs are able to inhibit the back
ground cleavage effectively. Ethidium bromide, a strong inter-
calator with minimal cytotoxicity, is also known to inhibit
background cleavage as well as DNA cleavage induced by other
drugs (34). Unlike ethidium bromide, AD280 and AD288, as
well as their C-14-substituted congeners AD 199 and AD 198,
induced PAB in cells and are cytotoxic in vitro or in vivo. The
observations taken together suggest that, following the treat
ment with these 3'-jY-alkylanthracyclines, a different mode of
drug-topoisomerase II-DNA interaction or perhaps a new type
of topoisomerase II inhibition occurs. It is also possible that
the analogues kill the cell by other mechanism(s), independently
of topoisomerase II.
Drug Structure and Cytotoxicity. The side chain C-14 su listi-

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 ANTHRACYCLINES AND TOPOISOMERASE
tution in AD32 and AD 143 is important for various biological
properties, including those connected with intracellular parti
tioning of the drug (reviewed in Ref. 7). The actual effectors of
topoisomerase II-related cytotoxicity are the C-14-unsubsti-
tuted metabolites of these compounds, mainly AD41. The
topoisomerase H-mediated DNA cleavage by 3'-W-acylanthra-
cyclines is comparable among the four C-14-unsubstituted an
alogues. However, AD38 and its C-14-substituted congener are
only marginally active in biological systems. While the 3'-N-
acetyl substitution of the two compounds has little effect on the
tests conducted in cell-free systems, it may diminish in some
way the cell kill efficacy. The C-13-OH forms of anthracyclines
such as AD92 are generally credited with lower cytotoxicity
relative to their parent drugs. Some of the 3'-A'-unsubstituted
(DXR, 4-dmxDNR) and the 3'-/V-alkyl analogues (for example,
AD 198, AD 199, AD280, and AD288) have shown a disparity
between their in vivo and in vitro effectiveness. In either case,
other topoisomerase II-unrelated mechanism(s) may participate
to a various degree in vivo and in vitro and contribute to the
overall cytotoxicity.
In conclusion, the study of 24 analogues of DXR or dauno-
mycin suggests that there are two domains of the anthracycline
molecule which may determine some of the biological proper
ties of the congeners. The first is localized at the C-14 position
of the chromophore Ring A. The side chain attached to the
chromophore, be it acyl or nonhydrolyzable ether, may prevent
the formation of a ternary complex drug-topoisomerase II-
DNA (7). Consequently, topoisomerase II is not stabilized and
the cleavable complex is not formed. The second domain,
important for anthracycline intercalation, is localized at the 3'-
nitrogen of the daunosamine moiety. The intercalative mode
seems to be advantageous for the drug interaction with topoi
somerase II-DNA complexes. While the daunosamine sugar of
the drug molecule is positioned along the minor grove of DNA
(44-46), the C-14 domain of the chromophore can be presented
for further interaction with binding sites of the enzyme mole
cule. With the increasing number of the intercalated drug
molecules, the interaction may reach a saturation level and
result in the inhibition of background cleavage. Since the back
ground inhibition is also present in C-14-substituted drugs
which do not stabilize the cleavable complex, these compounds
may exert topoisomerase II inhibition which differs from the
trapping of the cleavable complex.
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